Abstract
In this study, purification of recombinant human growth hormone (rhGH) with FLAG tag from E. coli inclusion bodies was described. The step-by-step process carried out was as follows: bacterial cells were suspended in lysis buffer and disrupted in a single sonication step. Then, inclusion bodies were isolated and solubilized in different buffers to obtain the best one which was found to be Tris buffer containing 2% deoxycholate with a satisfyingly adequate capacity to dissolve inclusion bodies at pH 12.5. In the third step, the proteins in solubilizing buffer were refolded by being diluted in refolding buffer. This was carried out with lowering the pH value to 8 using a direct dilution (to five volumes) process. Following a specific enterokinase cleavage for removing the tag, the rhGH was purified by ion-exchange chromatography. Following with the research, for the first time, a comparative study was performed on two weak ion-exchangers, namely DEAE and CM-Sepharose (two most commonly used resins), so as to investigate their pH dependence and resolution. DEAE-Sepharose at pH 8.25 exhibited the best overall performance and efficiency for rhGH purification as measured by either yield or purity. The overall process was reproducible and easy to scale up, making it a suitable choice for large-scale production of therapeutic proteins.
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